Improvement of the Performance of Targeted LC–MS Assays through

Oct 16, 2014 - Mass spectrometric-based quantification using targeted methods has matured during the past decade and is now commonly used in ...
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Technical Note pubs.acs.org/jpr

Improvement of the Performance of Targeted LC−MS Assays through Enrichment of Histidine-Containing Peptides Cédric Mesmin and Bruno Domon* Luxembourg Clinical Proteomics Center, Centre de Recherche Public de la Santé (CRP-Santé), Strassen 1445, Luxembourg S Supporting Information *

ABSTRACT: Mass spectrometric-based quantification using targeted methods has matured during the past decade and is now commonly used in proteomics. However, the reliability of protein quantification in complex matrixes using selected reaction monitoring is often impaired by interfering signals arising from coelution of nontargeted components. Sample preparation methods resulting in the reduction of the number of peptides present in the mixture minimizes this effect. One solution consists in the selective capture of peptides containing infrequent amino acids. The enrichment of histidinecontaining peptides via immobilized metal-ion affinity chromatography loaded with Cu2+ ions (IMAC-Cu) was applied in a quantitative workflow and found to be a simple and cost effective method for the reduction of sample complexity with high recovery and selectivity. When applied to a series of depleted human plasma digests, the method decreased nonspecific signals, resulting in a more precise and robust protein quantification. The method was also shown to be an alternative to HSA/IgG depletion during plasma protein analysis. This method, used in conjunction with recent improvements in the instrument’s peak capacity, addresses a bottleneck generally encountered in quantitative proteomics studies by providing the robustness and throughput required for the analysis of large sample series without compromising the number of proteins monitored. KEYWORDS: Mass spectrometry, targeted proteomics, enrichment, histidine, IMAC, depletion, selectivity

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parallel reaction monitoring8,9,13); second, by implementing an additional stage of gas-phase separation (e.g., MRM cubed,14 ion mobility15); third, through extensive protein/peptide fractionation (e.g., MuDPIT,16 isoelectric focusing,17 SISCAPA18). Furthermore, the mismatch between the wide range of protein concentrations in biological samples and the dynamic range of LC−MS analysis is another challenge, especially when dealing with biofluids such as serum and plasma.19,20 The isolation of a subproteome based on the enrichment of peptides containing infrequent amino acids (e.g., cysteine,21,22 methionine,23 or tryptophan24,25) was proposed to decrease sample complexity. The proteome will remain largely accessible for analysis, but the number of peptides per protein in the sample will be substantially reduced. Several implementations of this approach were previously reported, with most of them relying on multistep methods, including chemical derivatization of peptides followed by specific isolation on solid-phase supports.21−25 These methods, although effective, were mainly applied for discovery experiments due to reagent costs and lack of straightforward protocols.

INTRODUCTION Targeted proteomics approaches, based on liquid chromatography coupled to mass spectrometry (LC−MS), enable the systematic and precise quantification of peptides (and indirectly proteins) of interest required for experiments with samples from large patient cohorts.1−5 Although LC−MS methods are routinely used, as reflected by the growing number of publications in the biomarkers and systems biology areas, protein quantification by LC−MS in complex matrixes remains an analytical challenge. The complexity of biological samples, composed of numerous proteoforms, is amplified at the peptide level after proteolytic digestion, resulting in a very large number of peptide entities to be separated and characterized. The selectivity of the analytical system is restricted by its peak capacity, which is a limiting factor for the selected reaction monitoring (SRM) method commonly used for targeted LC− MS quantification.6−10 The two stages of mass filtering do not systematically resolve targeted from interfering ions, thereby reducing the accuracy and precision of the quantification. This limitation has been recognized, and data processing solutions have been developed to detect interfering signals postacquisition.6,11,12 In order to remedy the discrepancy between resolving power and sample complexity, several approaches have been tested: first, directly at the MS instrument level (e.g., © 2014 American Chemical Society

Received: August 4, 2014 Published: October 16, 2014 6160

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Trypsin Digestion of Plasma Samples

In contrast, histidine-containing peptide (His-peptide) enrichment through immobilized metal-ion affinity chromatography loaded with Cu2+ ions (IMAC-Cu)26−29 does not require chemical modification. It allows for the standardization of the assay using conventional stable isotope labeled peptides and relies on readily available inexpensive consumables. It thus represents an attractive fractionation method, although the effects of His-peptide enrichment on the results of targeted quantification have not been reported so far. In the present study, an optimized IMAC-Cu protocol was developed and evaluated in the context of targeted LC−MS analyses. The impact of the His-peptide capture on the interference in SRM measurements was studied and exemplified through comparison of the results from targeted protein quantification in a series of depleted human plasma samples. Furthermore, the method was investigated as an alternative to the classical HSA/IgG depletion to extend the dynamic range of plasma protein quantitative assays.

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For the nondepleted samples, 8 μL of plasma was diluted to a final volume of 100 μL with 0.1 M ammonium bicarbonate buffer containing 10% acetonitrile. Samples (either before or after HSA/IgG depletion) were heated at 99 °C for 10 min and, after cooling to 25 °C, RapiGest SF (Waters, Milford, MA, USA) was added to a final concentration of 0.1% (w/v). The proteins were reduced with dithiothreitol at a final concentration of 10 mM, for 50 min at 50 °C, and alkylated with iodoacetamide at a final concentration of 25 mM, for 30 min at 25 °C in the dark. Excess iodoacetamide was quenched by addition of dithiothreitol at a final concentration of 3 mM and further incubation for 30 min at 25 °C. A two-step digestion was performed by addition of sequencing grade-modified porcine trypsin (Promega, Madison, WI, USA) to a final enzyme/substrate ratio of 1:15 (w/w) and incubation for 16 h at 37 °C followed by a second addition of enzyme to a final enzyme/substrate ratio of 1:100 (w/w) and further incubation for 2 h at 37 °C. The peptide mixture obtained was subsequently subjected to solid-phase cleanup (no. WAT036820, Waters, Milford, MA, USA), and eluted with 50% acetonitrile. The eluate was aliquoted in four equal fractions, and dried in a vacuum centrifuge.

MATERIALS AND METHODS

Materials

The reagents used in this study were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Low-purity synthetic stable isotope-labeled (SIL) peptides with C-terminal 15 N- and 13C-labeled arginine and lysine residues were purchased from Thermo-Fisher (Ulm, Germany).

Histidine-Containing Peptide Enrichment Using IMAC-Cu Capture

His-peptide capture was performed using homemade, singleuse IMAC-Cu spin-columns. All steps described below were performed by centrifugation at 2000g for 80 s. Empty microspin columns (no. 89879/Pierce, ThermoFisher Scientific) were initially washed 2 times with 300 μL of a freshly prepared acetonitrile solution containing 25% (v/v) water. A 50 μL volume of a suspension of iminodiacetic acid derivatized sepharose beads was packed in the microspin column and washed twice as described above. Metal ions were loaded onto the column using 50 μL of a 30 mM copper(II) sulfate pentahydrate solution, and the activated column was washed two additional times as described above. The column was subsequently equilibrated 2 times with 300 μL of phosphate buffer (20 mM, pH 7.0) followed by 2 times with 300 μL of phosphate buffer (20 mM, pH 7.0) containing 50 mM NaCl and 0.05% (m/v) CHAPS. The protein digest (corresponding to ∼ 50 μg of initial protein) was resuspended in 50 μL of phosphate buffer (20 mM, pH 7.0) containing 50 mM NaCl and 0.05% (m/v) CHAPS and loaded onto the column. Unbound peptides were removed by successive washing steps, one time using 50 μL and three times with 300 μL of phosphate buffer (20 mM, pH 7.0) containing 50 mM NaCl and 0.05% CHAPS. In order to reduce the concentration of salts and CHAPS, a final washing step with 100 μL of phosphate buffer (20 mM, pH 7) was applied. The histidine-containing peptides were eluted from the column with 25 μL of a 25 mM ammonium formate solution containing 2.5% (v/v) acetonitrile adjusted to pH 2.5 using trifluoroacetic acid, which was repeated four times.

Preparation of Yeast Cell Lysate Digest

The yeast cell lysate digest was prepared as described earlier.8 The peptide mixture obtained was subsequently subjected to solid-phase cleanup (no. WAT036820, Waters, Milford, MA, USA). Peptides, eluted with 80% acetonitrile, were aliquoted with initial protein amounts corresponding to ∼50 μg (as determined by Bradford assay) and dried in a vacuum centrifuge. Human Plasma Samples

The blood samples used in this study were collected from 10 healthy volunteers. Prior to sample collection, informed consent forms approved by the Comité National d’Ethique de Recherche (CNER) were obtained from the patients. Blood samples were processed and stored according to the standard operating protocols of the Integrated BioBank of Luxembourg (IBBL). Depletion of Abundant Proteins from Plasma

The two most abundant plasma proteins, human serum albumin (HSA) and immunoglobulin G (IgG), were depleted using the multiple affinity removal spin cartridge HSA/IgG (no. 5188-8825, Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions. Briefly, for each depletion experiment, 25 μL of plasma was diluted to a final volume of 200 μL, filtered through a 0.22 μm spin filter (Agilent Technologies, Santa Clara, CA, USA), and loaded on the depletion column. After HSA/IgG removal, a buffer exchange of the flow-through fraction was performed using Vivaspin 3K spin-filter (Sartorius, Goettingen, Germany) to end up with proteins solubilized in 100 μL of 0.1 M ammonium bicarbonate buffer containing 10% acetonitrile. The protein concentrations were determined before and after depletion by Bradford assay.

LC Separation

Chromatographic separations were carried out on an Ultimate 3000 RSLC nano system (Thermo-Fisher Scientific). For each analysis, the sample was loaded into a C18 trapping column (Acclaim PepMap100, 2 cm × 75 μm i.d., 3 μm particule size, 100 Å porosity, Thermo-Fisher Scientific) and desalted for 10 min at 5 μL/min with an aqueous solution containing 0.05% (v/v) trifluoroacetic acid and 1% acetonitrile. The trapping 6161

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Table 1. Effect of Histidine-Containing Peptide (His-Peptide) Enrichment on Sample Complexitya

human

yeast

Full proteome His-peptides His-peptides retainedd His-peptides retainedd, Methionine free Full proteome His-peptides His-peptides retainedd His-peptides retainedd, Methionine free

no. peptide entitiesb

% peptide entities

no. proteins with SPc

% proteins with SP

avg. no. of SP per protein

620 360 164 069 134 989 100 937 176 528 38 579 31 480 23 536

100 26 22 16 100 22 18 13

19 970 18 824 18 455 17 493 6353 5811 5664 5349

100 94 92 88 100 91 89 84

28 8 7 6 27 6 5 4

a

Numbers were obtained through in silico tryptic digestion of the canonical protein sequences from the UniProtKB/Swiss-Prot database (ver. 2012_06). Only peptides without miss-cleavage were considered. bPeptide entities are here defined as distinct amino acid sequences with a number of residues being between 5 and 30. cSignature peptides (SP) are here defined as unique amino acid sequences in the considered proteome with a number of residues being between 5 and 30. dHis-peptides bearing one single histidine residue in position 2 or 3 from the N-terminus were shown not to be retained during His-peptide enrichment.

Analyses on a Triple Quadrupole Instrument

column was then switched in-line with a C18 analytical column (Acclaim PepMap100 RSLC 15 cm × 75 μm i.d., 2 μm particule size, 100 Å porosity, Thermo-Fisher Scientific), and peptide elution was performed by applying a mixture of solvent A (water containing 0.1% formic acid)/B (acetonitrile containing 0.1% formic acid) at 300 nL/min. For analysis using an LTQ-Orbitrap Velos mass spectrometer, the following steps were used: after 5 min at 2% solvent B, a linear gradient was started to reach 35% solvent B in 66 min, followed by a washing step (10 min at 90% solvent B) and an equilibration step (10 min at 2% solvent B). For analysis using a TSQ Vantage mass spectrometer, the following steps were used: after 3 min at 2% solvent B, a linear gradient was started to reach 35% solvent B in 48 min, followed by a washing step (10 min at 90% solvent B) and an equilibration step (10 min at 2% solvent B). Trapping and analytical columns were maintained at 35 °C.

Selected reaction monitoring analyses were performed using a TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher Scientific, San Jose, CA, USA) operating as previously described.9 Time-scheduled SRM methods were used to target the peptides of interest in 4 min retention time windows by monitoring from four to six transitions for each peptide within a cycle time of 2.5 s (a list of target peptides and transitions is provided in Supporting Information Table 1). A mixture of 15 synthetic, isotopically labeled peptides (no. 88321/Pierce retention time calibration mixture) was added to each sample at a concentration of 25 fmol/μL before analysis to allow offline retention time correction. Processing and Analysis of Data

The raw data were processed with either Xcalibur ver. 2.2 (Thermo-Fisher Scientific, San Jose, CA, USA) or Skyline ver. 2.5 (http://proteome.gs.washington.edu/software/skyline/). Automatic selection and delimitation of the chromatographic peaks by Skyline were manually reviewed. Dot-products were calculated between experimental and reference composite MS/ MS spectra as previously described.30 The MS/MS data obtained from LC−MS analysis on LTQOrbitrap Velos were searched against either human or Saccharomyces cerevisiae UniProtKB/Swiss-Prot database of canonical sequences (ver. 2011_08) using MASCOT (ver. 2.2) search algorithms through Proteome Discoverer 1.4 platform (Thermo-Fisher Scientific, Bremen, Germany). Trypsin was indicated as the protease with a maximum of two missed cleavages allowed. Carbamidomethylation of cysteine was specified as a static modification, and oxidation of methionine and cyclization of N-terminal glutamine were included as dynamic modifications. The mass error tolerance was set to 10 ppm for precursor ions and 0.01 Da for fragment ions. The data was also searched against a decoy database, and the results were used to estimate the false discovery rate within the Proteome Discoverer suite. Results were filtered to obtain a false discovery rate of 0.01.

Analyses on a LTQ-Orbitrap Velos Instrument

High-resolution/accurate mass analysis was performed using an LTQ-Orbitrap Velos mass spectrometer (Thermo-Fisher Scientific, San Jose, CA, USA) coupled to the nano-LC system described above through a dynamic nanoelectrospray source housing with uncoated silica emitters (12 cm length, 360 μm o.d., 20 μm i.d., 10 μm tip i.d., New Objective, Woburn, MA, USA). Ionization was obtained by applying a liquid junction voltage of 1500 V and a capillary temperature of 275 °C. Mass spectrometry analysis was carried out in data-dependent mode with full scan (300−2000 m/z) acquired with a resolving power of 60 000 at 400 m/z with the Orbitrap mass analyzer. The eight most intense precursor ions from a survey scan, with a signal exceeding 10 000, were selected for fragmentation using a 2.2 Da isolation window. Preview mode for FTMS master scans was disabled, whereas the injection time prediction and the monoisotopic precursor selection options were activated. Precursor ions with undetermined or 1+ charge were excluded from the data-dependent precursor selection process. Collision dissociation was performed in HCD mode using a normalized collision energy fixed at 35 and an analysis of the fragment ions at an Orbitrap resolving power of 7500 at 400 m/z. The option to trigger MS/MS events at the apex of the elution profile was enabled, and the correlation algorithm was used with a maximum area ratio to previous scan set at 0.9. Dynamic exclusion of 30 s with a 7 ppm mass window was used. The automatic gain control for full FT MS was set to 1 × 106 ions and for FT MS/MS was set to 3 × 104 ions, with maximum ion injection times of 500 and 1000 ms, respectively.

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RESULTS AND DISCUSSION

Characterization of the Histidine-Containing Peptide Enrichment Method

A peptide fractionation method intending to be applied for large-scale protein quantification should allow for a significant decrease in the number of peptide entities to be analyzed while 6162

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Figure 1. Reduction of sample complexity using histidine-containing peptide enrichment. (Left) Total ion current chromatogram of LC−MS analysis before (red) and after enrichment (blue). In both cases, an equivalent amount of initial protein (∼1 μg) was injected on-column. (Right) Mass spectrum averaged over a 30 s acquisition window at 45 min elution time (see left panel). The signal corresponding to histidine-containing and histidine-free peptides identified by database search is represented by stars and open circles, respectively.

recovered (yields of extraction below 5%), demonstrating the high selectivity of the capture toward His-peptides. Surprisingly, four His-peptides bearing a single histidine residue in position two or three from the N-terminus were not recovered. We hypothesized that this lack of retention is due to Cu2+ stripping from the solid support caused by a tighter complex formation of metal ions with both the N-terminal and imidazole nitrogen of these peptides than with the imminodiacetic from the stationary phase. The in silico simulation showed that the partial loss of these peptides has no significant impact on the proteome accessibility through His-peptide enrichment (Table 1). As expected, all of the other 72 His-peptides were recovered, albeit with variable yields of extraction. It should be emphasized that, when the IMAC-Cu capture is used in the context of stable isotope dilution experiments, the yields of extraction of the His-peptides will be corrected using the SIL internal standards spiked in before extraction, leading to an apparent recovery31 approaching 100% and to an unbiased quantification. The His-peptide enrichment was modeled using on the abundance of yeast proteins reported in the literature32 and their in silico trypsin digestion. It showed that one-sixth of the peptide molecules contained histidine residue(s), corroborating the LC−MS measurements (Figure 1 and Supporting Information Figure 1). It thus demonstrates the overall efficiency of the capture. The high selectivity of the Hispeptide enrichment was further confirmed by the identification after capture of 1630 His-peptides out of 1680 peptides (95%) (Supporting Information Figure 2) using a database search, supporting previous studies.27,29,33 Peptides identified correspond to proteins with abundances spanning 4 orders of magnitude, thus establishing the wide dynamic range of the method (Supporting Information Figure 2). The reproducibility of the His-peptide enrichment was evaluated by addition of a constant amount of a 61 SIL Hispeptide mixture into HSA/IgG-depleted human plasma digests originating from four healthy donors prior to IMAC-Cu capture. The analysis of the SIL peptides by SRM after His-

keeping the coverage of the proteome accessible to quantification. The reduction of the sample complexity through IMAC-Cu capture was simulated using the results of in silico trypsin digestion of both the yeast and human proteomes (Table 1). The data showed that more than 90% of the proteome remains accessible, with a number of peptide entities reduced by 4-fold. An average of 8 and 6 signature peptides per protein ensures confident analysis of the human and yeast proteomes, respectively. For quantitative analyses, it is common practice to omit peptides containing a methionine residue due to their chemical reactivity. His-peptides devoid of methionine residues still cover 88 and 84% of the human and yeast proteomes, respectively. An average of 6 and 4 signature peptides for each protein remains available to allow confident analysis of the human and yeast proteomes. To experimentally verify the effect of the His-peptide enrichment on the sample complexity, yeast digests were analyzed by LC−MS(/MS) before and after IMAC-Cu capture. The overall detected signal was significantly reduced after His-peptide enrichment, corresponding to a decrease in the number of ions analyzed, as illustrated in Figure 1. In the averaged spectrum recorded in a 30 s window centered at the 45 min elution time, all Hispeptides identified in the full-digest analysis were recovered and identified after IMAC-Cu capture, along with eight Hispeptides identified only after enrichment. In contrast, none of the peptides devoid of histidine residue(s) identified before enrichment was observed after IMAC-Cu capture, highlighting the selectivity of the method. Hence, His-peptide enrichment is an efficient method to decrease sample complexity, but to be implemented in routine quantitative workflows, the His-peptide enrichment must exhibit high analytical performance. To further confirm the high recovery and selectivity of the method, a study was carried out by SRM analysis on a set of 108 SIL peptides, composed of 76 His-peptides and 32 peptides devoid of a histidine residue, spiked into a yeast cell lysate digest before enrichment (Supporting Information Table 2). None of the 32 peptides devoid of a histidine residue were 6163

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Figure 2. Impact of histidine-containing peptide enrichment on the quality of SRM measurements in depleted human plasma. Signature peptides EVALDLSQHK (A) and WIVTAAHCVETGVK (B) of sulfhydryl oxidase 1 and coagulation factor 9, respectively, were monitored using six SRM transitions for the endogenous and stable isotope-labeled (SIL) forms. The SIL peptides were added to the sample prior to the extraction at a nominal concentration of 50 and 240 fmol/μL for panels A and B, respectively. The same amount of the initial plasma sample was injected oncolumn in both cases. The reference composite MS/MS spectra (gray bars) were acquired for the individual peptides without biological matrix under identical collision conditions. The similarity between reference and experimental spectra (red/blue bars) is indicated by the dot-product value (DP). (A) The traces corresponding to the fragment ions b3+, y8+2, y5+, y6+, y7+, and y8+ are represented in orange, light blue, purple, green, red, and dark blue, respectively. (B) The traces corresponding to the fragment ions y4+, y8+2, y9+2, y11+2, y7+, and y8+ are represented in green, orange, light blue, purple, red, and dark blue, respectively.

samples, were monitored as a proxy of the background noise. The signal-to-background noise (S/N) ratio was further determined for each transition for analyses performed both before and after His-peptide enrichment. Out of the 952 transitions monitored, 705 (74%) showed an increased S/N ratio after enrichment, of which 321 (34%) presented more than a 5-fold improvement, leading to an increase in the number of transitions that allowed peptide quantification (Supporting Information Table 2). The overall decrease of the background noise by simplification of the peptide mixture results in a more reproducible fragmentation pattern due to a considerable reduction of interference. This was demonstrated by addition of 61 SIL His-peptides, prior to IMAC-Cu capture, to HSA/IgG-depleted human plasma digests originating from 10 healthy donors. Both the SIL peptides and their endogenous counterparts were monitored by SRM before and after His-peptide enrichment.

peptide enrichment showed consistent signals across the four independent extractions, with reproducibility CVs below 20% for 56 out of the 61 peptides monitored, thus demonstrating the reproducibility of the method (Supporting Information Table 2). These results demonstrate the performance of the Hispeptide enrichment in decreasing sample complexity, which is, in turn, expected to improve the quality of targeted LC−MS assays due to the reduction of background signals. Impact of the Enrichment of Histidine-Containing Peptides on Quantitative Analysis

To evaluate the impact of a reduction in sample complexity on quantitative analyses results, a set of 238 endogenous Hispeptides, previously observed in HSA/IgG-depleted human plasma digests, were monitored by SRM. The transitions of the corresponding SIL peptides, not spiked into the plasma 6164

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Figure 3. Effect of histidine-containing peptide enrichment on the performance of SRM assays in depleted human plasma. Two signature peptides of (A) coagulation factor 9 and (B) complement component C8 beta chain were monitored using six SRM transitions for the endogenous and stable isotope-labeled (SIL) forms. The same amount of SIL peptides was added to each sample prior to enrichment at a nominal concentration of 70, 240, 440, and 310 fmol/μL for the peptides IIPHHNYNAAINK, WIVTAAHCVETGVK, YYAGGCSPHYILNTR, and GDYTLNNVHACAK, respectively. Areas under the curve (AUC) of the six transition chromatographic peaks were summed for each peptide. Relative signals were reported as the ratio of the endogenous peptide’s AUC to its respective SIL peptide’s AUC. The bar heights are averages, and error bars are standard deviations of two analytical replicates. The ratio between the relative signals of the two peptides of the same protein are reported for each sample. Chromatograms and spectra of sample 8 are presented in Figure 2B.

TGVK, with the removal of interfering signals for the y82+ and y7+ fragment ion transitions and in the y112+ channels of the endogenous and SIL forms, respectively. The improvement in the measurement quality leads to an increased robustness of the assays, as illustrated by the analysis of two signature peptides of coagulation factor 9 and complement component C8 beta chain in 10 human plasma samples (Figure 3). For both proteins, the His-peptide enrichment yielded more precise measurements, with interreplicate coefficients of variation ranging from 1 to 15% compared to coefficients of variation ranging from 1 to 48% without extraction. In addition, the consistency of the assays was enhanced by His-peptide enrichment. The relative signals obtained for two peptides originating from the same protein showed no coherence before extraction, with ratios varying from 1.7 to 3.3 and from 1.3 to 2.2 for coagulation factor 9 and complement component C8, respectively. In contrast, the steady values obtained for the ratios of relative signals after enrichment clearly reflect the proportional production of the two peptide signatures by trypsin digestion of the same protein. The improved precision and consistency of the assays are directly linked to the presence of variable interfering compounds before extraction that have been removed during the His-peptide enrichment, as previously illustrated for the peptide WIVTAAHCVETGVK, a surrogate of coagulation factor 9 (Figure 2B).

The similarity between the experimental and a reference composite MS/MS spectra (previously obtained by analysis of the SIL peptides under neat conditions) was assessed using the dot-product values. For more than two-thirds of the 1218 spectra analyzed, the similarity of the experimental fragmentation patterns with their references was improved by the Hispeptide enrichment (Supporting Information Table 2). This improvement, reflecting a decrease in interference, facilitates confirmation of the peptide’s identity and its proper quantification. As an example of the improvement of the data quality, the chromatograms and composite MS/MS spectra obtained in depleted human plasma digests for signature peptides of a candidate biomarker of breast cancer (sulfhydryl oxidase 1, EVALDLSQHK)34 and of one protein in the low microgram per milliliter concentration range (coagulation factor 9, WIVTAAHCVETGVK)35 are presented in Figure 2. The increase in the S/N ratio is clearly exemplified by the cleaner signals observed after His-peptide capture for the endogenous peptide EVALDLSQHK. Fragmentation patterns indicate a higher degree of similarity between experimental and reference spectra after IMAC-Cu capture, as indicated by the increase in the dot-product values. The removal of interference in the channels corresponding to the b3+ and y82+ fragment ions accounts for the improvement observed for these peptides. Similar results were obtained for the peptide WIVTAAHCVE6165

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Figure 4. Comparison of histidine-containing peptide enrichment and HSA/IgG depletion. Partial mass spectra observed in a 2 m/z window showed the signals of precursor ions resulting from different sample preparations. The peptides YCQVVCTYHPR, YLSDHSFLVSQGDR, LGDDLLQCHPAVK, and QQTHMLDVMQDHSR are proteotypic for factor XI, von Willebrand factor, vitamin K dependent protein C, and clusterin, respectively. The signal of the isotopic cluster of the precursor ions is presented in color, whereas nonspecific signal is shown in gray. (Top row) Digest of the full plasma sample directly analyzed by LC−MS. (Middle row) Digest of the full plasma sample subjected to histidine-containing peptide enrichment and analyzed by LC−MS. (Bottom row) Depletion of HSA/IgG performed for the same plasma sample followed by digestion and LC−MS analysis.

approaches, with the removal of 70−80% of the total peptide molecules, which was further confirmed by LC−MS analysis of digests of an undepleted plasma sample, a HSA/IgG-depleted sample, and a His-peptide-enriched fraction (Supporting Information Figure 3). For the latter two methods, the amount of the peptides loaded on the chromatographic column was thus increased 3- to 4-fold compared to that from the nontreated sample. As a consequence, the signals of the peptide precursor ions were increased in both cases, as exemplified in Figure 4 for the signature peptides LGDDLLQCHPAVK and QQTHMLDVMQDHFSR of vitamin K dependent protein C and clusterin, respectively. In contrast to HSA/IgG depletion, where a few hundred peptide entities were removed, the enrichment of His-peptides induces a dramatic decrease in the number of peptide entities analyzed (Table 1), leading to lower background and complexity. This point is illustrated in Figure 4, with the precursor ions of the signature peptides YCQVVCTYHPR and YLSDHSFLVSQGDR of factor XI and von Willebrand factor, respectively. This is of particular interest for analyses performed on trapping instruments, such as quadrupole-Orbitrap mass spectrometers operated in the selected ion monitoring or parallel reaction monitoring modes, since the accumulation of a more homogeneous population of ion leads to an increased dynamic range and sensitivity.30,38 The dynamic range of the plasmatic protein concentrations is not modified by the IMAC-Cu method, which yields few Hispeptides derived from HSA/IgG proteins present in the samples. The peptides derived from HSA detected after His-

Having a sample of lower complexity leads to less (unpredictable) interference, which is beneficial from a practical point of view, as high-intensity fragments can be selected more readily for SRM monitoring. In addition, lower sample complexity becomes of paramount importance in the context of the analysis of samples from a large cohort of patients, when matrix (and interference) may vary from individual to individual. Enrichment of Histidine-Containing Peptides as an Alternative to the Depletion of Abundant Proteins in Human Plasma

A commonly used approach to decrease the range of concentrations of human plasma proteins consists in the depletion of the most abundant components. Only a few proteins account for the large fraction of the total protein amount, and their removal allows the amount of peptides derived from low-abundance proteins to be increased, thus significantly expanding the range of detection.36 This benefit is, however, achieved at the expense of the cost and robustness of the assays. As a matter of fact, the depletion relies on expensive affinity reagents (e.g., antibodies, protein A/G), with no control over the co-removal of other compounds.37 These drawbacks remain a serious limitation to the use of abundant-protein depletion in large-scale studies such as biomarker evaluation. In this context, His-peptide enrichment represent an alternative and was compared to the classical plasma HSA/ IgG depletion. A simulation was conducted in silico using the reported concentrations of the most abundant plasma proteins.35 It showed a comparable effect of the two 6166

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Notes

peptide enrichment were separated properly, even if some tailing was observed, and they covered, in total, less than 5 min of the full 90 min chromatogram (i.e., 85 min are not affected). The presence of very abundant peptides does not affect a targeted assay. Furthermore, in our hands, no significant ion suppression for peptides coeluting with the HSA/IgG peptides was observed. His-peptide enrichment represents a real alternative to the commonly used protein depletion for routine stable isotope dilution proteomics studies, as it is simple, cost-effective, and scalable.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded through CORE (Lux-hPDQ) and PEARL (CPIL) grants from The Fonds National de la Recherche (FNR). The authors thank the Integrated BioBank of Luxembourg (IBBL) (Dr. Fay Betsou and Dr. Nikolai Goncharenko) and Dr. Guy Berchem (CHL) for providing samples, Katriina Sertamo and Dr. Kévin Demeure for assistance, Drs. Yeoun Jin Kim, Elodie Duriez, Jan Van Oostrum, and Sébastien Gallien for helpful discussions.

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CONCLUSIONS In this study, the enrichment of histidine-containing peptides through IMAC-Cu capture was optimized and applied to targeted protein quantification. These peptides may present favored ionization and particular fragmentation pathways (e.g., multicharged fragment ions) that are appropriate for SRM measurement, as documented in several reports.39−41 Because chemical derivatization is not required, the implementation of the method in existing isotope dilutionbased assays is straightforward, representing an advantage over other capture methods aimed at infrequent amino acid containing peptides. This simple enrichment method decreases sample complexity and reduces both interference and ion suppression effects in LC−MS analysis of complex mixtures, leading to improved precision and consistency of protein quantification. It exhibits high recovery and selectivity, which improves the assay’s sensitivity by allowing the injection oncolumn of a larger number of peptides as a consequence of the depletion of non-histidine-containing peptides. The enrichment of histidine-containing peptides in one single fraction is compatible with a high-throughput setup and does not sacrifice the analyte multiplexibility of the method. Thus, histidine-containing peptide enrichment does fill the gap between extensive fractionation and immuno-affinity enrichment methods in a cost-effective manner. Last, but not least, it represents a convincing alternative to plasma HSA/IgG depletion. This method, used in conjunction with recently developed LC−MS platforms of improved peak capacities, addresses a bottleneck generally encountered in quantitative proteomics studies by providing the robustness and throughput required for the analysis of a large sample series without compromising the number of proteins monitored.

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ABBREVIATIONS SRM, selected reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquid chromatography; SIL, stable isotope labeled; IMAC-Cu, immobilized metal-ion affinity chromatography loaded with copper II; Hispeptides, histidine-containing peptides

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ASSOCIATED CONTENT

S Supporting Information *

Table 1: SRM transitions monitored; Figure 1: Impact of the His-peptides enrichment on the total peptide concentration of yeast cell lysate digest; Figure 2: Dynamic range of the IMACCu capture; Table 2: Selected examples of peptide yields of extraction, signal to background noise values, and spectrum similarity values; Figure 3: Impact of His-peptides enrichment and HSA/IgG depletion on the total peptide concentration of human plasma digest. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +35226970717. 6167

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Journal of Proteome Research

Technical Note

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